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Introduction: Albeit that over 170 types of chemical modifications have been identified in RNAs, the biological functions of most of those decorations are still elusive. N7-methylguanosine (m7G), routinely occurring at the 5’ cap of mRNA or within tRNA and rRNA, also exists in internal mRNA. However, the “reader” proteins that recognize internal mRNA m7G and regulate the metabolism and fate of target mRNAs have yet to be identified. Here, we aim to identify the reader(s) of internal mRNA m7G and elucidate the biological function. Methods: To identify internal m7G reader(s), we utilized RNA-pull down, mass spectrometry (MS), RNA immunoprecipitation (RIP)-seq, and m7G-seq. To evaluate the interaction between QKI and G3BP1, we used Co-IP, immunofluorescence (IF) and proximity ligation assay (PLA). To investigate the function of QKI in stress granule (SG), we performed polyA & m7G RNA fluorescence in situ hybridization (FISH), triple quadrupole MS (QQQ-MS), and Ribo-seq. To assess the role of QKI in drug resistance, we carried out both in vitro cell survival assays and in vivo xenograft mouse model. Results: Our QQQ-MS results suggest cytoplasmic mRNA, in contrast to nuclei mRNA, are highly enriched with internal m7G modification. Quaking proteins (QKI), especially QKI7, can selectively recognize the internal mRNA m7G decoration in the cytosol of various cell types. Our high-throughput sequence data revealed over 1000 confident m7G-modified and QKI-binding RNA targets with a conserved motif, “GANGAN (N=A/U/G)”. Those transcripts are functionally enriched in the “pathways in cancer” and “Hippo signaling pathway”. More strikingly, internal m7G reader QKI7 directly interacts with the SG core protein G3BP1 and can shuttle a subset of m7G-modified transcripts into SG mRNA pool under oxidative stress condition. Additionally, we identified 314 confident m7G-modified & QKI7-binding & SG-enriched target genes in U2OS cells. Ribo-seq indicated that QKI7 modulates the translation efficiency of a set of m7G-modified transcripts via sequestering and silencing them in SGs. Moreover, in line with the observation that many chemotherapy drugs could trigger the assembly of SGs, QKI7 increases the sensitivity of cancer cells to drug treatment (e.g., doxorubicin) in vitro and in vivo in a m7G-dependent manner. Consistently, by analyzing the hepatocellular carcinoma (HCC) pharmacogenomic landscape study dataset, we found that HCC cell lines with higher QKI expression levels are more sensitive to a variety of chemotherapy or targeted therapeutic drugs. Conclusion: Our study identifies QKI as the first internal mRNA m7G reader, provides insights into the biological function of internal mRNA m7G modification in remodeling stress granules transcriptome, and highlights the therapeutic potential of targeting the QKI7/m7G axis in overcoming drug resistance. Citation Format: Zhicong Zhao, Lei Dong, Li Han, Yangchan Li, Jianhuang Xue, Keren Zhou, Wei Li, Xiaolan Deng, Ying Qing, Brandon Tan, Zhenhua Chen, Chao Shen, Andrew Small, Kitty Wang, Keith Leung, Zheng Zhang, Xi Qin, Qiang Xia, Rui Su, Jianjun Chen. Recognition of internal mRNA N7-methylguanosine by QKI shuttles transcripts into stress granules and modulates drug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5685.

Background: As the most prevalent internal decorations in mammalian mRNA, N6-methyladenosine (m6A) has been reported to be involved in many physiological and pathological processes, including acute myeloid leukemia (AML). METTL3 and METTL14, the well-recognized m6A methyltransferase complex, contribute to AML. METTL16 is a recently identified m6A methyltransferase that has been reported to deposit m6A in a few targets. While, unlike METTL3/14, the biological functions of METTL16 are largely unknown. Here, we explored the function and mechanism of METTL16 in AML pathogenesis and evaluated its therapeutic potential for AML treatment. Methods: We performed CRISPR-Cas9 screen to evaluate the dependency of METTL16 in AML cells. We created METTL16 knockout (KO) cells and conditional KO mice to evaluate its role in leukemogenesis and normal hematopoiesis. We employed bone marrow transplantation (BMT), xenograft, and AML patient-derived xenograft (PDX) models to determine its role in AML development and progression. To identify the targets of METTL16, we performed m6A-seq and RNA-seq, followed m6A-qPCR, CLIP-qPCR, in vitro methyltransferase assays and RNA stability assays. To examine the effect of METTL16 on branched chain amino acid (BCAA) metabolism, we performed metabolic profiling with 13C, 15N-leucine. Results: CRISPR-Cas9 screen showed METTL16 is one of the most essential genes for the survival of AMLs. The AML cells display more robust dependency on METTL16 than METTL3/14. We found METTL16 is highly expressed in AML patients compared to healthy controls. METTL16 KO significantly inhibited AML cell proliferation, promoted cell apoptosis and myeloid differentiation in vitro, which could be totally reversed by forced expression of wild-type METTL16, but not catalytic-dead mutant. METTL16 depletion dramatically inhibited AML progression and prolonged survival of recipient mice in the BMT, xenograft and PDX models. In addition, METTL16 is highly expressed in LSCs contrast to leukemic bulk cells and METTL16 KO significantly attenuates LSC self-renewal in vitro and in vivo. By contrast, the role of METLL16 is largely spared in normal hematopoietic cells. Via integrated analysis of m6A-seq data and RNA-seq data, we identified two bona fide targets of METTL16, BCAT1 and BCAT2, which encode two critical BCAA transaminases in BCAA biosynthesis pathway. METTL16 promotes the expression of BCAT1 and BCAT2 via an m6A dependent manner. Metabolomics with 13C, 15N-leucine tracing showed that METTL16 KO results in suppressed pools of TCA cycle intermediates, some non-essential amino acids and nucleotides. Conclusion: We uncover a tumor-promoting role of METTL16 in AML and LSC self-renewal via reprogramming BCAA metabolism, in which METTL16 functions as an m6A methyltransferase to regulate expression of BCAT1 and BCAT2. Our data suggest that METTL16 is an attractive target for AML therapy. Citation Format: Li Han, Lei Dong, Keith Leung, Zhicong Zhao, Yangchan Li, Ying Qing, Jianhuang Xue, Chao Shen, Zhenhua Chen, Lei Gao, Kitty Wang, Keren Zhou, Wei Li, Brandon Tan, Zheng Zhang, Xi Qin, Rui Su, Xiaolan Deng, Jianjun Chen. METTL16 drives leukemogenesis and maintains leukemia stem cell self-renewal via reprogramming BCAA metabolism [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3617.